U.S. patent application number 12/891327 was filed with the patent office on 2011-04-07 for antihuman baff antibody.
This patent application is currently assigned to TSUTOMU TAKEUCHI. Invention is credited to Tsutomu Takeuchi, Keiko Yoshimoto.
Application Number | 20110081351 12/891327 |
Document ID | / |
Family ID | 35999997 |
Filed Date | 2011-04-07 |
United States Patent
Application |
20110081351 |
Kind Code |
A1 |
Takeuchi; Tsutomu ; et
al. |
April 7, 2011 |
ANTIHUMAN BAFF ANTIBODY
Abstract
An antibody against a peptide having an amino acid sequence
AVQGPEETVT QDC (expressed in single letter amino acid code) as
represented by SEQ ID: NO. 1 corresponding to the 134- to
146-positions in human BAFF (B cell activating factor belonging to
the TNF family) protein which is preferably a monoclonal antibody;
a method of producing the above antibody; a medicinal composition
containing the antibody; utilization of the antibody; and a method
of screening an inhibitory effect or an activating effect on BAFF
with the use of the antibody.
Inventors: |
Takeuchi; Tsutomu; (Tokyo,
JP) ; Yoshimoto; Keiko; (Tokyo, JP) |
Assignee: |
TAKEUCHI; TSUTOMU
TOKYO
JP
YOSHIMOTO; KEIKO
TOKYO
JP
KOWA COMPANY, LTD.
NAGOYA-SHI
JP
|
Family ID: |
35999997 |
Appl. No.: |
12/891327 |
Filed: |
September 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11661260 |
Feb 27, 2007 |
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PCT/JP2005/015696 |
Aug 30, 2005 |
|
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12891327 |
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60605516 |
Aug 31, 2004 |
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Current U.S.
Class: |
424/139.1 ;
435/7.92; 436/501; 530/387.9 |
Current CPC
Class: |
G01N 33/564 20130101;
A61P 37/06 20180101; A61P 3/10 20180101; A61P 5/00 20180101; A61P
29/00 20180101; G01N 2500/00 20130101; A61P 19/02 20180101; G01N
33/74 20130101; A61K 2039/505 20130101; C07K 16/241 20130101; G01N
33/68 20130101; A61P 37/00 20180101; A61P 37/02 20180101; C07K
14/70575 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 436/501; 435/7.92 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; G01N 33/53 20060101
G01N033/53; A61P 37/06 20060101 A61P037/06 |
Claims
1. An antibody against a peptide having an amino acid sequence
comprising AVQGPEETVT QDC (expressed in single letter amino acid
code) in the 134- to 146-positions in human BAFF (B cell activating
factor belonging to the TNF family) protein.
2. The antibody according to claim 1, wherein the antibody is a
monoclonal antibody.
3. A process for producing an antibody against a peptide having an
amino acid sequence comprising at least AVQGPEETVT QDC (expressed
in single letter amino acid code), from an antibody-producing cell
in an animal, by sensitizing the animal with an antigen comprising
the peptide.
4. The process according to claim 3, wherein the peptide is a
peptide comprising an amino acid sequence consisting of AVQGPEETVT
QDC (expressed in single letter amino acid code).
5. The process according to claim 3 or 4, wherein the antigen
further comprises a hapten.
6. The process according to claim 3 or 4, wherein the animal is a
mouse, rat, hamster, guinea pig, chicken or rabbit.
7. A pharmaceutical composition comprising the antibody according
to claim 1 or 2 and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition according to claim 7, wherein the
pharmaceutical composition is to prevent and treat an autoimmune
disease.
9. The pharmaceutical composition according to claim 8, wherein the
autoimmune disease is SLE, RA, SS, autoimmune diabetes, AIDS, or an
autoimmune disease accompanied by B-cell activation.
10. A diagnostic agent for an autoimmune disease, which comprises
the antibody according to claim 1 or 2.
11. The diagnostic agent according to claim 10, wherein the
autoimmune disease is SLE, RA, SS, autoimmune diabetes, AIDS, or an
autoimmune disease accompanied by B-cell activation.
12. The diagnostic agent according to claim 10, wherein the
diagnosis is carried out by ELISA.
13. A method of detecting or quantifying BAFF in a sample, which
comprises adding the antibody according to claim 1 or 2 to a sample
and measuring BAFF bound to the antibody.
14. The method according to claim 13, wherein BAFF is human
BAFF.
15. The method according to claim 13, wherein the method of
measuring BAFF bound to the antibody is carried out by ELISA.
16. A method of screening the inhibiting or activating action of a
test substance on BAFF, which comprises adding a test substance to
a sample containing BAFF and measuring a change, upon addition of
the test substance, in the amount of BAFF by the antibody according
to claim 1 or 2.
17. The method according to claim 16, wherein BAFF is human
BAFF.
18. The method according to claim 16, wherein the method of
screening the inhibiting action on BAFF is screening of a
prophylactic/therapeutic agent for autoimmune diseases.
19. Use of the antibody according to claim 1 or 2 for producing a
pharmaceutical composition for prophylaxis/therapy of autoimmune
diseases.
20. Use of the antibody according to claim 1 or 2 for producing a
composition for diagnosis of autoimmune diseases.
21. Use of the antibody according to claim 19, wherein the
autoimmune disease is SLE, RA, SS, autoimmune diabetes, AIDS, or an
autoimmune disease accompanied by B-cell activation.
22. A method of preventing or treating an autoimmune disease, which
comprises administering an effective amount of the antibody
according to claim 1 or 2 to a patient with an autoimmune disease
or a patient with a risk of an autoimmune disease.
23. The method according claim 22, wherein the autoimmune disease
is SLE, RA, SS, autoimmune diabetes, AIDS, or an autoimmune disease
accompanied by B-cell activation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/661,260, filed on Feb. 27, 2007 which is a 371 of
International Application No. PCT/JP2005/015696 filed on Aug. 30,
2005 which is based upon and claims the benefit of priority from
the prior Application No. 60/605,516, filed on Aug. 31, 2004, the
entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a novel antihuman BAFF
antibody, preferably an antihuman BAFF monoclonal antibody, and a
process for producing the same. In addition, the present invention
relates to a pharmaceutical composition for prophylaxis and therapy
of an autoimmune disease such as systemic lupus erythematosus
(SLE), chronic rheumatoid arthritis (RA), Sjogren's syndrome (SS),
autoimmune diabetes, AIDS, or an autoimmune disease accompanied by
B-cell activation, which includes use of the antibody, preferably
the monoclonal antibody, a diagnostic agent and diagnostic method
comprising the same, and a prophylactic/therapeutic method for an
autoimmune disease which includes administering an effective amount
of the antibody. Further, the present invention relates to a method
of quantifying BAFF and a method of screening a substance having an
inhibiting action or an activating action on BAFF, which includes
use of the antibody, preferably the monoclonal antibody.
BACKGROUND ART
[0003] It is known that BAFF (B cell activating factor belonging to
the TNF family) is produced and secreted from T cells,
monocytes/macrophages, dendritic cells and the like and regulates
such as B-cell differentiation, activation, survival rate via three
types of receptors on B cells (Moore et al., Science, 285, 260-263
(1999)).
[0004] Human BAFF is a transmembrane form protein comprising 285
amino acids. There is a structural characteristic of trimer
formation such as the presence of a cytoplasmic domain of 46 amino
acids, an extracellular domain of 218 amino acids, and two
N-glycosylation sites in its amino acid sequence. It is estimated
that an extracellular domain of 152 amino acids from C-terminal is
cleaved with a protease of Furin family and released in a soluble
form. The amino acid sequence of human BAFF initially named
Neutrokine a was disclosed as SEQ ID NO: 1 or 2 in publication of
International Patent Application WO98/18921. Other names of human
BAFF such as Kay, TNFSF13B, Blys, TALL-1, THANK and zTNF4 are also
known.
[0005] BAFF-R, TACI (transmembrane activator and calcium modulator
and cyclophilin ligand interactor), and BCMA (B cell maturation
antigen) are known as BAFF receptors. BAFF-R and BCMA are expressed
mainly in B cells, and TACI is expressed in B cells and activated T
cells.
[0006] The physiological action of BAFF lies in regulation of
B-cell differentiation, activation, survival rate and the like as
described above, and is increasingly revealed in recent years to
participate in pathologic condition. That is, it is reported that a
mouse expressing BAFF in excess shows SLE-like symptoms such as
increase in peripheral blood B cells, enlargement of lymph nodes
and spleen, increase in IgG level in serum, antinuclear antibody
production, deposition of immune complex in the kidney, albuminuria
and nephritis (Mackay et al., J. Exp. Med., 190, 1697-1710, (1999),
and Khare et al., Proc. Natl. Acad. Sci. USA 97, 3370-3375,
(2000)). It was further reveled that this mouse also shows SS-like
symptoms such as inflammation of salivary gland and destruction of
salivary gland with advancing age (Groom et al., J. Clin. Invest.,
109, 59-68, (2002)). An increase of BAFF level in serum in patients
suffering from SLE, RA and SS is also reported (Groom et al., J.
Clin. Invest., 109, 59-68, (2002); Zhang et al., J. Immunol., 166,
6-10, (2001); and Cheema et al., Arthritis Rheum., 44, 1313-1319,
(2001)), and there are also many reports such as higher BAFF level
in synovial fluid than in serum in patients suffering from RA
(Cheema et al., Arthritis Rheum., 44, 1313-1319, (2001)),
expression of BAFF in salivary gland-infiltrating leukocytes in
patients with SS (Groom et al., J. Clin. Invest. 109, 59-68,
(2002)), correlation between serum BAFF level in patients suffering
from SLE and immunoglobulin or anti-ds DNA antibody (Zhang et al.,
J. Immunol. 166, 6-10, (2001)) and correlation between BAFF in
patients suffering from RA and rheumatoid factor (Cheema et al.,
Arthritis Rheum., 44, 1313-1319, (2001)).
[0007] From these facts, it can be said that measurement of BAFF in
serum or tissue (for example, synovial fluid in patients suffering
from RA) not only in patients with autoimmune diseases such as SLE,
RA and SS but also in patients before onset of these diseases is
useful for prehension of the progress of clinical state, that is,
for diagnosis. Autoimmune diseases such as SLE, RA and SS can also
be prevented and treated by inhibiting the functions of BAFF.
[0008] For measurement of a protein such as BAFF, a method of using
an antibody capable of recognizing the protein is general. As
antibodies against BAFF, there are actually some commercial
products including monoclonal and polyclonal antibodies (for
example, antihuman monoclonal antibody (Catalog Number: MAB124)
manufactured by R&D Systems, goat antihuman BAFF polyclonal
antibody (Catalog Number: SC-5743) manufactured by Santa Cruz
Biotech, mouse antihuman BAFF monoclonal antibody (Catalog Number:
ALX-804-128-C100) manufactured by ALEXIS) and the like. These
antibodies are produced against full-length BAFF or its C-terminal
amino acid sequence as antigen, do not exhibit high specificity for
human recombinant BAFF in Western blotting, and cannot be
satisfactory in respect of detection limit in ELISA, and none of
such known antibodies satisfy high-sensitivity diagnosis.
[0009] The method of inhibiting functions of BAFF to prevent or
treat immune diseases such SLE, RA and SS includes a method of
suppressing or inhibiting production of BAFF by suppressing
expression of BAFF gene, a method of inhibiting BAFF receptor by a
BAFF receptor antagonist or the like, and a method of inhibiting
functions of BAFF itself by an anti-BAFF antibody or the like. For
example, it is known that the clinical test, by Human Genome
Sciences Ltd., of antihuman BAFF (BLyS.TM.) monoclonal antibody
(development name: LymophoStat-B.TM.) in SLE or RA patients is
advanced to phase-2 clinical test in the US, but the possibility
thereof as a therapeutic agent is not necessarily satisfactory and
there is demand for development of an antibody having a further
useful working effect as a prophylactic or therapeutic agent.
DISCLOSURE OF INVENTION
[0010] The object of the present invention is to provide an
antihuman BAFF antibody with higher sensitivity, which can be used
in diagnosis of autoimmune diseases. By using such antihuman BAFF
antibody, many autoimmune diseases can be more efficiently
prevented and treated, and the present invention provides such a
pharmaceutical composition and a prophylactic/therapeutic method of
using the same. By using such antihuman BAFF antibody, there can
also be provided a method of screening a human BAFF inhibiting or
activating agent.
[0011] Accordingly, the present inventors made extensive study, and
as a result, found that when a novel monoclonal antibody (referred
to hereinafter as 4H4) prepared by using an antigen having KLH
(keyhole limpet hemocyanin) bound to 13 amino acids (SEQ ID NO: 1)
as hapten corresponding to a region, in the vicinity of a membrane,
of an extracellular domain in the amino acid sequence of human
BAFF, is used as a detection antibody in ELISA for detection of
human BAFF, detection of surprisingly high sensitivity attaining a
detection limit of 0.5 ng/mL is made feasible, and the present
invention was thereby completed.
[0012] That is, the present invention relates to an antibody,
preferably a monoclonal antibody, against a peptide having an amino
acid sequence comprising AVQGPEETVT QDC (expressed in single letter
amino acid code) in the 134- to 146-positions in human BAFF (B cell
activating factor belonging to the TNF family) protein.
[0013] Further, the present invention is to provide for producing
the antibody of the invention. Specifically, the present invention
relates to a process for producing the antibody against a peptide
having an amino acid sequence comprising at least AVQGPEETVT QDC
(expressed in single letter amino acid code), from an
antibody-producing cell in an animal, by sensitizing the animal,
preferably a nonhuman animal, with an antigen containing the
peptide.
[0014] The present invention provides various applications or uses
of the antibody of the invention described above.
[0015] That is, the present invention relates to a pharmaceutical
composition comprising the antibody of the invention described
above and a pharmaceutically acceptable carrier, preferably a
pharmaceutical composition for preventing and treating autoimmune
diseases. Further, the present invention relates to a method of
preventing or treating autoimmune diseases, which includes
administering an effective amount of the antibody of the invention
to patients suffering from autoimmune diseases or patients with a
risk of autoimmune diseases. The present invention also relates to
use of the antibody of the invention for producing a pharmaceutical
composition for prophylaxis/therapy of autoimmune diseases or a
diagnostic composition for autoimmune diseases.
[0016] The present invention also relates to a diagnostic agent for
autoimmune diseases comprising the antibody of the invention as
well as a diagnostic method using the same.
[0017] Further, the present invention relates to a method of
detecting or quantifying BAFF in a sample, which includes adding
the antibody of the present invention to a sample and measuring
BAFF bound to the antibody.
[0018] The present invention also relates to a method of screening
the inhibiting action or activating action of a test substance on
BAFF, which includes adding a test substance in a sample comprising
BAFF and measuring, by the antibody of the present invention, a
change in the amount of BAFF upon addition of the test
substance.
BRIEF DESCRIPTION OF DRAWINGS
[0019] FIG. 1 is a drawing of recombinant human BAFF (manufactured
by Chemicon) was detected by conventional Western blotting with the
antihuman BAFF monoclonal antibody (4H4) of the invention and a
control rabbit antihuman BAFF polyclonal antibody (Chem)
manufactured by Chemicon.
[0020] FIG. 2 is a graph showing a standard curve prepared with
recombinant human BAFF (manufactured by Chemicon) by ELISA
established by using 4H4.
[0021] FIG. 3 is a graph showing the effect of 4H4 on IgG
production induced by stimulating healthy person- or SLE
patient-derived PBL with anti-CD3 antibody.
[0022] FIG. 4 is a graph showing the effect of 4H4 on IFN.gamma.
production induced by stimulating healthy person- or SLE
patient-derived PBL with anti-CD3 antibody.
[0023] FIG. 5 is a graph showing the effect of 4H4 on INF.alpha.
production induced by stimulating healthy person- or SLE
patient-derived PBL with anti-CD3 antibody.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024] The antibody of the present invention is characterized by
being produced by using an antigen comprising a peptide comprising
the following amino acid sequence (see SEQ ID NO: 1) :
[0025] AVQGPEETVT QDC
in the 134- to 146-positions corresponding to a region, in the
vicinity of a membrane, of an extracellular domain in the amino
acid sequence of human BAFF comprising 285 amino acids.
[0026] Usual antibodies are produced by using, as antigen, an N- or
C-terminal amino acid sequence of a protein. This is because an
amino acid sequence in the vicinity of the center of a protein is
sometimes located stereoscopically in the protein interior and is
thus sometimes unsuitable as antigen. The N- or C-terminal side of
a protein is a stereoscopically exposed structure in many cases,
and it is well-known that an antibody against the N- or C-terminal
side has sufficient sensitivity in many cases. Accordingly, an
antihuman BAFF antibody has been conventionally produced against an
amino acid sequence of the N- or C-terminal side. However, the
present inventors found that the antihuman BAFF antibody thus
produced does not necessarily have sufficient sensitivity. The
reasons for this are not fully clarified, but it is expected that
the N- or C-terminal side of human BAFF, unlike usual protein, has
a structure which is not rendered sufficiently exposed. That is,
the reason that the antihuman BAFF antibody produced against an N-
or C-terminal side amino acid sequence does not necessarily have
sufficient sensitivity is considered attributable to a
3-dimensional structure unique to human BAFF, but is not fully
elucidated. It is expected that elucidation of the 3-dimensional
structure of human BAFF in the future will clarify the details.
[0027] The antibody of the present invention is characterized by
using, as antigen, a peptide including an amino acid sequence in an
almost central part of human BAFF, and the fact that the amino acid
sequence in such a central part is suitable as the antigen must be
surprising and is estimated to be attributable to a 3-dimensional
structure unique to human BAFF protein.
[0028] The antibody of the present invention is characterized by
using the above peptide as antigen, and the antibody may be a
polyclonal or monoclonal antibody, preferably a monoclonal antibody
from the viewpoint of specificity.
[0029] The antibody of the present invention comprises a naturally
occurring antibody obtained by immunizing a nonhuman mammal with
the antigen, according to a usual manner of producing an antibody,
a recombinant chimera monoclonal antibody and recombinant human
monoclonal antibody (CDR-grafted antibody) which can be produced by
using recombinant DNA techniques, and a human antibody which can be
produced by a human antibody-producing transgenic animal and the
like. The monoclonal antibody comprises monoclonal antibodies
having any isotypes of IgG, IgM, IgA, IgD and IgE. IgG or IgM is
preferable.
[0030] More specifically, the antihuman BAFF monoclonal antibody of
the invention is produced according to an existing general
production method by immunizing a mammal, preferably a mouse, rat,
hamster, guinea pig, rabbit, chicken, cat, dog, pig, goat, horse or
bovine, more preferably a mouse, rat, hamster, guinea pig or
rabbit, with an antigen having KLH bound to 13 amino acids (SEQ ID
NO: 1) as hapten corresponding to a region, in the vicinity of a
membrane, of an extracellular domain in the amino acid sequence of
human BAFF, if necessary together with Freund's adjuvant, thereby
yielding cells (cells from such as the spleen, lymph node, bone
marrow or tonsilla, preferably B cells from the spleen) producing
the antibody, then preparing hybridomas by using the cells and
cells of the bone marrow (myeloma cells) not having an ability to
produce an antibody, cloning the hybridomas and selecting them for
a clone producing a monoclonal antibody showing specific affinity
for the antigen used in immunization of the mammal by immunoassays
(ELISA or the like).
[0031] Specifically, the monoclonal antibody of the present
invention can be produced in the following manner. That is, a
mouse, rat, hamster, guinea pig, chicken or rabbit, preferably a
mouse, rat or hamster (including a transgenic animal,created as to
produce an antibody derived from other animal, for example, a human
antibody-producing transgenic mouse) is subjected to
immune-sensitization by injecting or transplanting an antigen
having KLH (keyhole limpet hemocyanin) bound to 13 amino acids (SEQ
ID NO: 1) as hapten corresponding to a region, in the vicinity of a
membrane, of an extracellular domain in the amino acid sequence of
human BAFF, if necessary together with Freund's adjuvant, once to
several times subcutaneously, intramuscularly, intravenously,
intraperitoneally or via a foot pad. Usually, the mammal is
immunized once to four times every about 1 to 14 days after first
immunization, and from the immune-sensitized animal about 1 to 5
days after final immunization, antibody-producing cells can be
obtained and used to yield a clone producing the monoclonal
antibody, according to the method described above.
[0032] Preferably, the monoclonal antibody of the present invention
can be produced as follows. That is, 100 .mu.L of 1 mg/mL aqueous
solution of the above antigen peptide in physiological saline,
together with Freund's complete adjuvant, is emulsified by
sonication and used in intraperitoneally immunizing a mouse
(Balb/c. 6-week-old). 100 .mu.L of 1 mg/mL aqueous solution of the
antigen peptide in physiological saline and Freund's complete
adjuvant, which were emulsified by sonication, are used as booster
for additional immunization 2 weeks after first immunization,
followed by additional immunization twice at 2-week intervals, and
thereafter, a clone producing the monoclonal antibody can be
obtained by the method described above.
[0033] Preparation of the hybridoma secreting a monoclonal antibody
can be carried out according to the method of Kehler and Milstein
(Nature 256, 495-497 (1975)) or a modification thereto. That is,
the hybridoma is prepared by subjecting antibody-producing cells
contained in the spleen, lymph node, bone marrow or tonsilla,
preferably the spleen, obtained from a mammal immune-sensitized as
described above, to cell fusion with a myeloma cell not having an
ability to produce an autoantibody, derived from a mammal,
preferably a mouse, rat, guinea pig, hamster, rabbit or human, more
preferably a mouse, rat or human.
[0034] The myeloma cell usable in cell fusion includes, for
example, mouse-derived myeloma P3/X63-AG8.653 (653; ATCC No.
CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/O-Ag14
(Sp2/0, Sp2), PAI, FO and BW5147, rat-derived myeloma
210RCY3-Ag.2.3, human-derived myeloma U-266AR1, GM1500-6TG-A1-2,
UC729-6, CEM-AGR, D1R11 and CEM-T15.
[0035] Screening of a hybridoma clone producing the monoclonal
antibody can be carried out by culturing the hybridoma in for
example a microtiter plate and then measuring, by RIA or enzyme
immunoassay such as ELISA, the reactivity of a culture supernatant
in a well showing proliferation, with the immune antigen used in
the immune sensitization of a mouse described above. Production of
the monoclonal antibody from the hybridoma can be carried out in
vitro, or in vivo in ascites fluid in a mouse, rat, guinea pig,
hamster or rabbit, preferably a mouse or rat, more preferably a
mouse, and the monoclonal antibody can be isolated from the
resulting culture supernatant or the ascites fluid of the mammal.
In the case of in vitro culture, the hybridoma is proliferated,
maintained and stored depending on various conditions such as the
properties of the cell cultured, the object of test and study, and
the culture method, and every nutrient medium such as a known
nutrient medium used in producing the monoclonal antibody or a
nutrient medium derived from a known basal medium can be used.
[0036] The basal medium includes, for example, low-calcium medium
such as Ham's F12 medium, MCDB153 medium or low-calcium MEM medium
and high-calcium medium such as MCDB104 medium, MEM medium, D-MEM
medium, RPMI1640 medium, ASF104 medium or RD medium, and the basal
medium can contain, for example, serum, hormone, cytokine and/or
various inorganic or organic substances if necessary. Isolation and
purification of the monoclonal antibody can be carried out by
subjecting the culture supernatant or the ascites fluid to
precipitation with saturated ammonium sulfate, precipitation with
euglobulin, a caproic acid method, a caprylic acid method,
ion-exchange chromatography (DEAE or DE52) or affinity column
chromatography on an anti-immunoglobulin column or protein A
column. From the hybridoma, a gene encoding the monoclonal antibody
is cloned and used to produce, by using the transgenic animal
producing technique, a transgenic rabbit, goat, sheep or pig in
which the antibody encoding gene was integrated in the endogenous
gene, and from milk of the transgenic animal, the monoclonal
antibody derived from the antibody gene can be obtained in a large
amount (Nikkei Science, pp. 78-84, April issue, 1997).
[0037] The antihuman BAFF monoclonal antibody of the invention
obtained as described above has a surprising feature that 0.5 mg/mL
detection limit can be attained as shown in the Examples described
later, and thus it is possible to realize not only a highly
sensitive method for diagnosing an autoimmune disease but also a
prophylactic/therapeutic method for an autoimmune disease and a
method of screening a human BAFF inhibiting or activating agent. In
the Examples, the antihuman BAFF monoclonal antibody of the
invention can be utilized as the antibody, an antibody fragment and
a derivative thereof. The antihuman BAFF monoclonal antibody of the
present invention can also be used in purification of BAFF derived
from cells or blood. The human BAFF protein and a derivative
thereof purified by using the antihuman BAFF monoclonal antibody of
the present invention can be utilized as a reagent or a
pharmaceutical preparation such as B-cell activator. Further, the
antihuman BAFF monoclonal antibody of the present invention, a
fragment of the antibody and a derivative thereof can be utilized
to make an image of BAFF protein by techniques known in the art,
such as immunostaining.
[0038] The pharmaceutically acceptable carrier in the
pharmaceutical composition of the present invention includes an
excipient, a diluent, an extender, a disintegrating agent, a
stabilizer, a preservative, a buffer agent, an emulsifier, an
aromatic substance, a coloring agent, a sweetener, a viscous agent,
a taste corrective, a solubilizing agent or other additives. By
using one or more of such carriers, it is possible to prepare
pharmaceutical compositions in the form of tablets, pills, powder,
granules, an injection, a solution, capsules, a troche, an elixir,
a suspension, an emulsion or a syrup. The pharmaceutical
composition of the present invention can be parenterally
administered. The form of the parenteral administration includes
eye drops and nose drops in addition to an injection formulated in
a usual manner, a suppository and pessary for enteric
administration, comprising the antibody of the present
invention.
[0039] The dose of the active ingredient in the pharmaceutical
composition of the present invention varies depending on the age,
sex, weight and condition of the patient, the therapeutic effect,
administration method and treatment time; usually, the active
ingredient can be administered to an adult in an amount in the
range of 1 .mu.g to 1000 mg, preferably 10 .mu.g to 500 mg, for
each administration. However, the dose varies depending on various
conditions, and thus a dose lower than the above dose maybe
sufficient in some cases, or a dose higher than the above dose
maybe necessary in other cases. For example, an injection can be
produced by dissolving or suspending the antibody in a nontoxic
pharmaceutically acceptable carrier such as physiological saline or
distilled water for injection at a concentration of 0.1 .mu.g
antibody/mL carrier to 10 mg antibody/mL carrier.
[0040] The injection thus produced can be administered in a dose of
1 .mu.g to 100 mg, preferably 50 .mu.g to 50 mg, for each
administration, per body kg once to several times per day to a
patient in need of treatment. The administration form can be
exemplified by medically suitable administration forms such as
intravenous injection, subcutaneous injection, intradermal
injection, intramuscular injection and intraperitoneal injection.
The administration form is preferably intravenous injection. The
injection can also be prepared as a suspension or emulsion with a
non-aqueous diluent (for example, propylene glycol, polyethylene
glycol and vegetable oils such as olive oil and alcohols such as
ethanol) depending on the case. Sterilization of such injections
can be carried out by filter sterilization (that is, through a
bacteria-retaining filter) or with a sterilizer or through
irradiation. That is, a germ-free solid composition is produced by
lyophilization and the like can be dissolved in germ-free distilled
water for injection or other solvent just before use.
[0041] The pharmaceutical composition of the present invention is
useful for prophylaxis/therapy of autoimmune diseases such as
systemic lupus erythematosus (SLE), chronic rheumatoid arthritis
(RA), Sjogren's syndrome (SS), autoimmune diabetes, AIDS or an
autoimmune disease accompanied by B-cell activation.
[0042] The diagnostic agent of the present invention comprises the
antibody of the present invention, and contains various reagents
binding specifically and highly sensitively to BAFF protein in a
sample thereby enabling measurement of a formed complex. The
diagnostic composition of the present invention comprises the
diagnostic agent of the present invention or the diagnostic agent
and a carrier. The composition of the present invention can also be
labeled so as to enable measurement of the antibody. For such
labeling, a usual method of using a radioactive element or a
fluorescent substance can also be used.
[0043] In the present invention, the method of diagnosing an
autoimmune disease can examine, for example, the progress of an
autoimmune disease by establishing an ELISA system with the
antihuman BAFF monoclonal antibody of the invention and measuring,
in this ELISA system, the concentration of BAFF in serum or tissue
collected from a subject such as a patient with an autoimmune
disease, as described later in the Examples. The diagnostic method
can also be utilized in monitoring for knowing the therapeutic
effect or in prediction of prognosis.
[0044] In the present invention, the method of detecting or
quantifying BAFF can be carried out by adding the antibody of the
present invention to a sample and measuring BAFF bound to the
antibody. In the method of measuring BAFF bound to the antibody of
the present invention, a wide variety of known techniques of
detection or quantification by the antigen-antibody reaction can be
used, and for example, the ELISA method can be used.
[0045] BAFF in the diagnostic method or the detection or
quantification method according to the present invention is
preferably human BAFF.
[0046] The prophylactic/therapeutic method for autoimmune diseases
in the present invention can realize the prophylaxis/therapy of
autoimmune diseases by administering the antihuman BAFF monoclonal
antibody of the invention together with a pharmaceutically
acceptable carrier to patients in need of the prophylaxis/therapy
of autoimmune diseases.
[0047] The method of screening a human BAFF inhibiting or
activating agent in the present invention can be realized by
establishing an ELISA system with the antihuman BAFF monoclonal
antibody of the invention and then measuring, in this ELISA system,
the BAFF binding activity of a test sample with a BAFF
receptor-expressing cell or a BAFF receptor protein or its fragment
and a derivative thereof, as described later in the Examples.
Screening of a human BAFF production inhibiting or promoting agent
can be carried out by contacting a test sample with a human
BAFF-producing cell and measuring BAFF produced by the cell, by a
conventional immunological technique known in the art, such as
ELISA.
[0048] The antihuman BAFF monoclonal antibody of the present
invention is extremely excellent in practical utility because of
its surprising feature that the detection limit of 0.5 mg/mL can be
attained as shown in the Examples shown later. The fact that the
antibody obtained from a peptide comprising an amino acid sequence
in the vicinity of in the center of the membrane protein can be an
antibody very excellent in specificity and affinity is also beyond
expectation because of the unique feature of BAFF.
[0049] The antibody of the present invention is characterized by
the site used as antigen against it, is extremely excellent in
specificity and affinity (sensitivity), and is capable of realizing
not only practical method for diagnosing an autoimmune disease but
also prophylactic/therapeutic methods for autoimmune diseases,
further a method of screening a human BAFF inhibiting or activating
agent.
[0050] An excellent pharmaceutical composition and diagnostic agent
for autoimmune diseases can be provided by using the antibody of
the present invention.
EXAMPLES
[0051] Hereinafter, the present invention is described in more
detail by reference to the Examples, but the present invention is
not limited by the Examples.
Example 1
Preparation of Antihuman BAFF Antibody
[0052] 13 amino acids corresponding to a region, in the vicinity of
a membrane, of an extracellular domain in 285 amino acids of BAFF
shown in SEQ NO: 2 in the Sequence Listing were selected, then
conjugated with KLH by the MBS method, and used as antigen. 100
.mu.L of 1 mg/ml aqueous solution of the antigen peptide in
physiological saline and Freund's complete adjuvant were formed
into an emulsion by sonication and then used in intraperitoneally
immunizing a mouse (Balb/c, 6-week-old). After 2 weeks, 100 .mu.L
of 1 mg/ml of an aqueous solution of the antigen peptide in
physiological saline and Freund's complete adjuvant, which had been
emulsified by sonication, were used as booster for additional
immunization, followed by additional immunization twice at 2-week
intervals. Two months after the first immunization, the spleen was
excised, and lymphocytes were separated in RPMI 1640 medium
(containing penicillin and streptomycin). The separated lymphocytes
were fused with mouse bone marrow-derived myeloma cell P3U1 strain
by the polyethylene glycol (PEG) method to prepare hybridoma cells.
The hybridoma cells were suspended in a feeder cell-containing HAT
medium, pipetted to a 96-well plate (Greiner) and cultured for 15
days. A culture supernatant was recovered from the wells in which
the hybridoma cells had been cultured, and antibody-producing cells
reactive with the antigen peptide were selected by ELISA
(enzyme-linked immunosorbent assay). That is, first, 50 .mu.L of 10
.mu.g/mL antigen peptide was put to each well of the 96-well plate,
adsorbed onto the bottom at 4.degree. C. overnight and blocked with
100 .mu.L of 2% BSA/PBS at 37.degree. C. for 2 hours. Each well was
reacted at 4.degree. C. overnight with 100 .mu.l supernatant of the
hybridoma cells and then reacted at 37.degree. C. for 1 hour with a
1000-fold dilution of HRP-labeled anti-mouse IgG and colored with
orthophenylene diamine as substrate. After the reaction was
terminated with 50 .mu.L of 2 N sulfuric acid, each well was
measured for absorption at 492 nm, and hybridomas showing an
absorption of 1.0 or more were selected and cloned by limiting
dilution.
[0053] A mouse (Balb/c) to which 0.5 mL pristane had been
intraperitoneally administered before 7 days and before 3 days, and
the selected hybridoma cells were injected intraperitoneally to the
mouse, and about 10 days later, the ascites fluid was collected.
The collected ascites fluid was left at room temperature for 30
minutes, then left at 4.degree. C. overnight, centrifuged at 15
Krpm for 10 minutes to recovery a supernatant from which a mouse
IgG fraction was separated and purified through a protein
A-Sepharose column.
[0054] By the method described above, a hybridoma cell strain
producing the antihuman BAFF antibody (4H4 whose isotype is IgG1),
as well as the antibody (4H4), was obtained. The resulting 4H4, and
a control rabbit antihuman BAFF polyclonal antibody (AB16530: shown
as Chem in FIG. 1) manufactured by Chemicon, were used in detection
of recombinant human BAFF (manufactured by Chemicon) by the usual
Western blotting method. As a result, a 17-KDa band corresponding
to the soluble human BAFF was confirmed as shown in FIG. 1.
Example 2
Establishment of ELISA
[0055] A 96-well plate was coated at 4.degree. C. overnight in a
volume of 1 .mu.g/well with a rabbit antihuman BAFF polyclonal
antibody (AB16530, manufactured by Chemicon) as primary antibody.
Each well was washed 3 times with PBS containing 0.05% Tween 20,
and then Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added
in a volume of 150 .mu.L/well and reacted at 37.degree. C. for 2
hours. Each well was washed 3 times with PBS containing 0.05% Tween
20, and 50 .mu.L of sample and 50 .mu.l (8 ng/mL) of biotin-labeled
4H4 were added and reacted at room temperature for 2 hours. Each
well was washed 3 times with PBS containing 0.05% Tween 20, and 50
.mu.L of a 1000-fold dilution of streptavidin-labeled HRP (horse
radish peroxide) diluted with PBS containing 0.05% Tween 20 was
added and reacted at room temperature for 30 minutes. Each well was
washed 5 times with PBS containing 0.05% Tween 20, and then 50
.mu.L of TMB One Solution (manufactured by Clonetech) was added and
reacted for 5 minutes at room temperature, then 50 .mu.L of 1N HCl
was added, and the each well was measured for absorbance at 450 nm
with a plate reader (manufactured by Perkin Elmer). As a standard
substance, recombinant human BAFF (manufactured by Chemicon) was
used to prepare a standard curve.
[0056] As a result, establishment of ELISA system showing an
excellent linear relationship at a BAFF concentration of from 25
ng/mL to 0.2 ng/mL was confirmed. Particularly, while a
concentration in the range of 2 ng/mL to 0.5 ng/mL cannot be
detected by conventional antihuman BAFF antibody, such reliable
detection at this concentration reveals that the diagnosis of
autoimmune diseases by using the antihuman BAFF monoclonal antibody
of the present invention or the screening of a BAFF inhibiting or
activating agent is very useful.
Example 3
Action on Human PBL
[0057] Blood was collected from a healthy person and patients
diagnosed as having SLE, and a lymphocyte layer was separated and
collected therefrom by gravity centrifugation with Ficoll, to give
Peripheral Blood Lymphocytes (PBLs). PBLs were suspended in
RPMI1640 medium containing 10% FBS (Fetal bovine serum), and
anti-CD3 antibody diluted at 10 .mu.g/mL with PBS was put to a
24-well culture plate and adsorbed at 4.degree. C. overnight onto
the bottom, and PBLs were inoculated at 5.times.10.sup.5
cells/well. Simultaneously, 4H4 was added at a final concentration
of 10 .mu.g/mL, followed by culture for 4 days or 7 days at
37.degree. C. in 7% CO.sub.2 in a CO.sub.2 incubator. The culture
supernatant was collected and measured by sandwich ELISA method
using monoclonal antibodies (IgG, primary antibody, manufactured by
BDPharmingen, Cat. No. 555784; secondary antibody (biotin-labeled),
manufactured by BDPharmingen, Cat. No. 555785; IFN.gamma., primary
antibody, manufactured by BDPharmingen, Cat. No. 554698; secondary
antibody (biotin-labeled), manufactured by BDPharmingen, Cat. No.
554550; INF.alpha., primary antibody, manufactured by BDPharmingen,
Cat. No. 551220; and secondary antibody (biotin-labeled),
manufactured by BDPharmingen, Cat. No. 554511) reacting
specifically with IgG, IFN.gamma. and TNF.alpha. in the culture
supernatant, respectively.
[0058] The results are shown in FIGS. 3 to 5. Production of IgG
(FIG. 3), IFN.gamma. (FIG. 4) and TNF.alpha. (FIG. 5) induced by
stimulation with anti-CD3 antibody (all of which are shown in the
left (open) column in the graph) from PBL derived from a healthy
person and an SLE patient was regulated by addition of 4H4 (all of
which are shown in the right (solid) column in the graph).
Particularly, it was revealed that the induction of production of
IgG, IFN-.gamma. and TNF-.alpha. by anti-CD3 antibody is higher in
patients than in healthy person, and the degree of suppression of
production thereof by 4H4 is also high. Particularly, it was
revealed that the production of TNF.alpha. by this antibody is
significantly suppressed (t-test). Promotion of such production is
considered to participate considerably in formation of clinical
state, and suppression of such production is estimated to be a new
attempt at therapy of autoimmune diseases such as SLE. Accordingly,
it was suggested that the antihuman BAFF monoclonal antibody of the
present invention is very useful for development of therapeutic
agents for autoimmune diseases.
INDUSTRIAL APPLICABILITY
[0059] The present invention is industrially applicable because it
provides an extremely specific and highly sensitive antibody to
BAFF, particularly human BAFF, which comes to be revealed to have
relationship with autoimmune diseases such as systemic lupus
erythematosus (SLE), chronic rheumatoid arthritis (RA), Sjogren's
syndrome (SS), autoimmune diabetes, AIDS or an autoimmune disease
accompanied by B-cell activation, and is not only useful for
therapy, prophylaxis and diagnosis of autoimmune diseases, but also
provides a method of screening a new substance useful for therapy
and prophylaxis of autoimmune diseases.
Sequence CWU 1
1
2113PRTHomo sapiens 1Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln
Asp Cys1 5 102285PRTHomo sapiens 2Met Asp Asp Ser Thr Glu Arg Glu
Gln Ser Arg Leu Thr Ser Cys Leu1 5 10 15Lys Lys Arg Glu Glu Met Lys
Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30Arg Lys Glu Ser Pro Ser
Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45Ala Ala Thr Leu Leu
Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60Ser Phe Tyr Gln
Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg65 70 75 80Ala Glu
Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95Ala
Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105
110Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn
115 120 125Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val
Thr Gln 130 135 140Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro
Thr Ile Gln Lys145 150 155 160Gly Ser Tyr Thr Phe Val Pro Trp Leu
Leu Ser Phe Lys Arg Gly Ser 165 170 175Ala Leu Glu Glu Lys Glu Asn
Lys Ile Leu Val Lys Glu Thr Gly Tyr 180 185 190Phe Phe Ile Tyr Gly
Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205Gly His Leu
Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220Ser
Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu225 230
235 240Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu
Gly 245 250 255Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln
Ile Ser Leu 260 265 270Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys
Leu Leu 275 280 285
* * * * *